This invention relates to a novel progestin-regulated gene. The protein or polypeptide encoded by this gene appears to have a novel enzymatic activity that may be useful as a readily detectable marker for progestin-responsiveness.
The sex steroid hormone progesterone has two major roles in mammalian physiology. First, progesterone is involved in preparing the uterus for implantation of the fertilized ovum. Second, progestins have proliferative and differentiating effects on mammary epithelium (1, 2). Progesterone is essential for lobuloalveolar development and preparation for lactation: when ovulation is established progesterone, produced by the corpus luteum, stimulates growth of the lobuloalveolar structures and during pregnancy promotes branching of the ductal system and differentiation of alveolar cells into secretory cells ready for milk production. The importance of progestin in these processes is clearly illustrated in progesterone receptor (PR) knockout mice, which fail to develop lobuloalveolar structures (3). Progestins may also have a role in regulating cell proliferation in the human breast. Mitotic activity in breast epithelium varies in a cyclic manner through the menstrual cycle and a role for progesterone in this process is suggested by observations that levels of this hormone and epithelial cell proliferation are both maximal during the late secretory phase (4). Some breast tumours retain progesterone responsiveness and the use of high doses of synthetic progestins are recognised endocrine therapies for PR-positive breast cancers, since in this scenario progestins have an antiproliferative effect (5). Progestins also have predominantly growth inhibitory effects on human breast cancer cell lines in vitro, although under certain conditions they may stimulate growth (2, 6 and references therein). Mechanistic studies have clearly defined both a stimulatory and inhibitory effect of progestins on breast cancer cell cycle progression (6) but the functional consequences of these effects in vivo remain to be defined. This is of considerable importance given the wide spread pharmacological usage of progestins in oral contraceptives and in hormone replacement therapy.
The mechanisms underlying the biological effects of progestins in the normal breast and in breast cancer are only partially understood. Progestin action is mediated primarily via the PR, which upon activation by ligand binding interacts with gene promoter sequences containing progesterone responsive elements (PREs) to regulate gene transcription. Very few mammalian genes have been described that are directly regulated by progestins in this manner: examples include c-jun (7), cfos (8), fatty acid synthetase (FAS) (9), PR (10, 11), and uteroglobin (12, 13). While progestin action ultimately involves changes in the levels of large numbers of mRNAs and proteins, many of these require intermediary de novo protein synthesis. Furthermore, specific genes that mediate the proliferative effects of progestins are likewise poorly defined. Thus much remains to be learned about genes induced as an acute response to progestin treatment and their role in mediating progestin effects on cell proliferation and differentiation.
Several known progestin-regulated genes can be classed as those whose functions are important in differentiation effects mediated by progestin. Examples include FAS (9), alkaline phosphatase (14) and lactate dehydrogenase (15). While these are probably not involved in the proliferative effects of progestin a number of progestin-related genes related to steroid and growth factor action might contribute to these effects at least indirectly. Examples include estrogen receptor (16), PR (11), retinoic acid receptors (17), epidermal growth factor receptor (6, 18), prolactin receptor (19), insulin-like growth factors xcex1 and xcex21 (6, 8, 23, 24), 17xcex2-hydroxysteroid dehydrogenase (25) and insulin-like growth factor binding proteins 4 and 5 (26). However, evidence that these gene products are direct mediators of the stimulatory and inhibitory effects of progestins remains to be determined. Potentially of more interest are progestin-regulated genes with known roles in cell cycle control i.e. c-myc, c-fos (6, 8), c-jun (7) and cyclin D1 (27). Progestin induction of c-myc and cyclin D1 is closely related to changes in cell cycle progression (6, 27). While the timing of the induction of c-myc mnRNA, evident after 30 min. of progestin treatment, suggests a potential direct effect of progestins, the slower induction of cyclin D1 MRNA which is maximal at 6 hours could result from the prior induction of other genes that are the primary and specific targets of progestins. Given the established central role for cyclin D1 in steroid and growth factor regulation of breast cancer cell cycle progression (27-30) identification of progestin regulated genes which control cyclin D1 gene expression might link progestin action to the cell cycle.
Using serum-free conditions, studies in this laboratory have shown that T-47D human breast cancer cells which were stimulated to grow with insulin, undergo a transient increase in cell cycle progression in response to progestins. with an increased rate of progression through G1 and a transient increase in the S-phase population. These cells complete a round of replication and thereafter become growth arrested early in G1 phase.
The present invention arose out of a study using this model system to identify novel progestin regulated genes involved in early cell cycle stimulatory responses to progestin or other aspects of progestin action in human breast cancer cells. RNA extracted from T-47D cells grown under serum-free conditions and treated with the synthetic progestin ORG 2058 for 3 hours was used as the template for cDNA synthesis and analysis by the differential display technique (mRNA fingerprinting) (31). Several candidate PCR fragments were identified by this method and characterisation by sequence and Northern analysis of some of these led to the identification and characterisation of a clone. PRG1, which appears to represent a novel progestin-regulated gene.
Thus, in a first aspect, the present invention provides an isolated DNA molecule comprising a nucleotide sequence substantially corresponding or, at least,  greater than 80% (more preferably,  greater than 90%) homologous to any one of the nucleotide sequences shown at:
(i) FIG. 2B from nucleotide 1 to 3018;
(ii) FIG. 2B from nucleotide 1 to 470;
(iii) FIG. 2B from nucleotide 141 to 3018: and
(iv) FIG. 2B from nucleotide 470 to 2103.
Preferably. the isolated DNA molecule is of human origin. More preferably, the isolated DNA molecule is of human kidney cell or breast cancer cell origin, and/or encodes a protein normally expressed in human kidney cells, breast tissue or tumour cells.
The isolated DNA molecule may be incorporated into plasmids or expression vectors. which may then be introduced into suitable bacterial, yeast and mammalian host cells. Such host cells may be used to express the polypeptide encoded by the isolated DNA molecule.
The predicted amino acid sequence of the polypeptide encoded by PRG1 shows substantial homology (xcx9c70%) with human liver 6-phosphofructo-2-kinase/fructose 2.6 bisphosphatase (PFK-2/FBPase-2) and it is postulated that the protein encoded by PRG1 may have activities similar to this bifunctional enzyme.
Thus, in a second aspect, the present invention provides a polypeptide in a substantially pure form, said polypeptide comprising an amino acid sequence substantially corresponding to that shown at FIG. 2B or an enzymatic portion thereof.
Preferably, the polypeptide of the second aspect is full length.
The polypeptide of the second aspect may be used to raise monoclonal or polyclonal antibodies which may be used, for example, in affinity purification processes or in various ELISA type assays.
Thus, in a third aspect, the present invention provides an antibody specific to the polypeptide of the second aspect.
As will be seen hereinafter, PRG1 appears to be directly regulated by progestin. PRG1 may, therefore, provide a useful marker for progestin-responsiveness in a subject. For example, as a marker of breast tumour responsiveness to progestinsxe2x80x94i.e. high levels may indicate that the tumour is responsive to progestins and could be sensitive to progestin therapy. PRG1 may also be a useful prognostic marker since hormonally responsive tumours often have a better prognosis (i.e. patients have longer disease-free survival and overall survival). Thus, levels of PRG1 MRNA present in isolated cells or tissue samples may be assessed by DNA or RNA probes or primers in hybridisation assays or PCR analysis. Suitable probes and primers, which are preferably of a length greater than 10 nucleotides, are to be considered as forming part of the present invention. Alternatively, the level of PRG1 polypeptide may be assessed through the use of the abovementioned antibodies. However, the postulated enzymatic activity of the PRG1 polypeptide provides the potential for a more convenient assay wherein the level of PRG1 polypeptide would be determined by assessment of enzyme activity.
Thus, in a fourth aspect, the present invention provides as assay for assessing progestin-responsiveness in a subject comprising the steps of;
(i) isolating cells or tissue from said subject; and
(ii) detecting the presence of a polypeptide comprising an amino acid sequence substantially corresponding to that shown at FIG. 2B.
Preferably, the polypeptide detection step involves providing a substrate for said polypeptide, said substrate normally converted by said polypeptide to a readily detected product.
In some circumstances, it may be preferred to expose the isolated cells or tissue to progestin or an agonist compound and, subsequently, determine whether the progestin or agonist compound has induced the production of PRG1 polypeptide.
The postulated enzyme activity of the PRG1 polypeptide also suggests that this polypeptide has an involvement in cell cycle (growth) regulation and is likely to be involved in control of glycolytic/gluconeogenic/lipogenic pathways not only in progestin target tissues but in a wide range of different tissue types. Indeed, since PRG1 appears to be expressed in tissues with a probable low fraction of proliferating cells it is unlikely that the function of the PRG1 polypeptide is restricted to growth regulation. More likely, PRG1 is more generally involved in glycolytic/gluconeogenic/lipogenic control. This may be of particular significance as the other related human enzyme PFK-2/FBPase-2, which has an established important role in glycolytic control, has only a limited tissue distribution (e.g. liver). Thus, the administration of PRG1 polypeptide may be of significant therapeutic value particularly for treatment of diabetes, obesity or other disorders of energy metabolism. Therapeutic amounts are likely to be similar to normal endogenous levels (which will vary from tissue to tissue) or may be at significantly higher levels, depending on the level and type of activity desired.
Alternatively, the enzyme activity of the PRG1 polypeptide could be regulated by pharmacological means for the treatment of proliferative disorders, such as malignant or non-malignant hyperproliferative disease (e.g. breast and other cancers, and dermatological diseases). Further, administration of PRG1 polypeptide may be of therapeutic value in the control of reproductive function.
More specifically, the enzyme activity of the PRG1 polypeptide could be regulated by;
synthetic compounds, either stimulatory or inhibitory (i.e. agonists or antagonists),
ribozymes specific for PRG1 (i.e. to down-regulate endogenous PRG1 activity), and
gene therapy using expression vectors or oligonucleotides or other delivery systems (e.g. viral) containing a nucleotide sequence encoding PRG1 sense (i.e. to augment endogenous PRG1 polypeptide levels and activity) or antisense (i.e. to down-regulate endogenous PRG1 levels and activity).
Agonist or antagonist compounds could be identified by their ability to inhibit/stimulate the enzyme activity of PRG1 polypeptide. For example, screening assays could be conducted to identify compounds that modulate 6-phosphofructo-2-kinase activity by measuring the rate of production of fructose-2,6-biphosphate from fructose-6-phosphate (assaying fructose-2,6-biphosphate by its ability to activate pyrophosphate-dependent 6-phosphofructo-1-kinase from potato tubers) (58). Alternatively, screening assays could be conducted to identify compounds that modulate fructose-2,6-biphosphatase activity by measuring [32P] release from [2-32P] fructose-2,6-bisphosphate (59). Such screening assays may be performed using in vitro systems such as dissolved pure PRG1 polypeptide or a whole cell lysate of cells expressing PRG1.
Such agonist and antagonist compounds may include compounds which influence enzymatic activity by a number of mechanisms such as the alteration of substrates to the enzyme""s active site(s), either by acting as alternative substrates or by binding to PRG1 polypeptide to alter its structure, or by influencing processes involved in the activation of the PRG1 polypeptide (e.g. phosphorylation of regulatory domains).
Results provided hereinafter strongly suggest that induction of PRG1 by progestin is a direct transcriptional effect of ligand-activated PR on a putative progestin-regulatory element(s) (PRE) located within the PRG1 gene,
Thus, in a fifth aspect, the present invention provides an isolated DNA molecule comprising a progestin-regulatory element (PRE) derived from a DNA molecule according to the first aspect of the invention.
The DNA molecule of the fifth aspect may be used as a controlling element for PRG1 (and potentially for other genes containing similar promoter elements), for novel therapeutics to control gene expression or could be utilised in DNA constructs designed to express RNA/protein sequences in response to progestins (e.g. progestin-inducible plasmids) which could be useful as research tools for studying gene expression in cell lines. The DNA molecule of the fifth aspect could also be of use in gene therapy directed to progestin-responsive tissues e.g. breast, uterus.
The term xe2x80x9csubstantially correspondsxe2x80x9d as used herein in relation to the nucleotide sequence is intended to encompass minor variations in the nucleotide sequence which due to degeneracy in the DNA code do not result in a change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.
The term xe2x80x9csubstantially correspondingxe2x80x9d as used herein in relation to amino acid sequence is intended to encompass minor variations in the amino acid sequence which do not result in a decrease in biological activity of the encoded protein. These variations may include conservative amino acid substitutions. The substitutions envisaged are: G,A,V.I,L.M: D, E; N,Q; S,T; K,R,H; F.Y,W,H; and P,Nxcex1-alkalamino acids.
The invention will now be further described with reference to the following non-limiting example and accompanying figures.